Cholera is a human diarrheal disease caused by the bacterium Vibrio cholerae. This disease remains a health threat for the majority of the world, causing thousands of deaths every year. Our laboratory has shown that the alternate sigma factor, sigma54, contributes to the virulence of V. cholerae. Sigma54 regulates multiple cellular processes, including flagellar and chemotaxis gene transcription. Both motility and chemotaxis have been shown by various means to contribute to V. cholerae virulence, but the mechanism(s) linking these behaviors to virulence has been elusive. We have shown that the activity of one of the sigma54-dependent activators, FlrC, regulates enhanced intestinal colonization as well as flagellar synthesis. Flagellar synthesis, in turn, regulates the expression of a polysaccharide (VPS) necessary for biofilm formation. VPS expression is modulated by another sigma54-dependent activator, VpsR. VpsR is also necessary for virulence factor expression and intestinal colonization. Thus sigma54 appears to contribute to flagellar synthesis, chemotaxis, biofilm formation, and virulence, in part through two different (54-dependent activators, FlrC and VpsR. This proposal will focus on the contributions of these two sigma54-dependent activators to virulence and biofilm formation, as well as address the contribution of chemotaxis, a CT54-dependent phenomenon, on V. cholerae virulence. Experiments are designed to: 1. Identify the factors that regulate FlrC activity, as well as the gene(s) regulated by FlrC that contribute to intestinal colonization. 2. Characterize the role of chemotaxis in V. cholerae virulence, focusing primarily on methyl-accepting chemoreceptors (MCPs), and 3. Analyze the contribution of VpsR to VPS expression and virulence factor expression. These experiments are designed to illuminate how sigma54 controls the virulence and environmental persistence (i.e., biofilm formation) of V. cholerae through two activators, FlrC and VpsR, and through chemotaxis. The results should lead to a deeper understanding of virulence mechanisms of V. cholerae, and facilitate the discovery of novel antimicrobial therapies that can prevent cholera.